In order to define the organization of rabbit immunoglobulin (Ig) genes and the mechanisms that regulate their expression during lymphoid cell differentiation, we have combined our earlier approaches of immunochemistry and immunogenetics with molecular biology. Thus, we isolated rabbit Ig mRNAs of defined genetic origins, constructed and sequenced cDNA probes, and are using these probes to study expressed mRNAs and genomic DNAs prepared from genetically defined rabbits. Poly (A)+ RNAs were prepared from spleens of hyperimmunized rabbits of a variety of allotypes and cell free synthesis of allotype-specific Gamma, Mu and light chains demonstrated by immunoprecipitation. Clones carrying cDNAs encoding Kappa light chains of b5, b9 and bas allotypes as well as heavy chain VHal, VHa2, Gamma 12, 14 and Mu 80 allotypes have been constructed, identified and sequenced. Some kappa chain variable region sequences from different allotypes are more similar to each other than are the sequences encoding the constant regions of b4, b5, b9 and bas "allelic" types (79-89% nucleic acid and 60-78% amino acid sequence homologies). The DNA sequences of these same molecules are remarkably homologous (93-96%) in their 3 feet untranslated (3 feet UT) regions. The high conservation of 3 feet UT regions enables us to detect Kappa light chain mRNAs from rabbits of different b allotypes on northern and dot blots. Our sequencing of DNAs encoding different rabbit Ig allotypes has allowed us to prepare and characterize allotype-specific probes. These will be used to determine the nature of mRNAs produced by purported latent-allotype producing cells cultured and manipulated in vitro, or collected from living animals. These and other studies using the probes are aimed toward achieving our goals of defining the organization and regulated expression of rabbit Ig genes in molecular terms and understanding the evolutionary origins and functional significance of complex rabbit allotypes.